Os autores gostariam de agradecer ao Dr. Scott Rizaldy no Hospital Monte Sinai em Toronto, Ontário, pelo seu dom da principal Rac1 flox / flox fibroblastos embrionárias de camundongos. Este trabalho foi financiado por subvenções de funcionamento a NJ do Canadian Institutes of Health Research (CIHR, MOP 2009-93526) e de Ciência Natural e Pesquisa de Engenharia Council of Canada (NSERC, RG 327372-06). SH e MW são suportados pelo CGSMA e CGS M-prêmios de CIHR e NSERC, respectivamente. Wills Steve Hawley e Melanie contribuíram igualmente para este papel.

Células descongelamento <ol><li> Obtenção de fibroblastos de rato congelado embrionárias (MEFs) (gerado anteriormente a partir de culturas primárias individuais de embriões) de nitrogênio líquido. </li><li> Células descongelar por imersão frasco em 37 ° C de água com movimentos circulares até conteúdo esteja completamente descongelado. </li><li> Adicionar 9ml de DMEM que foi suplementado com 10% de soro fetal bovino e 1% de penicilina-estreptomicina a uma placa de cultura de células 10 centímetros. </li><li> Adicione a suspensão de células descongeladas gota a gota, à placa, rock a placa suavemente para dispersar as células e colocar a placa em 37 ° CO C, 5% 2 incubadora durante a noite. Note-se que se as células são sensíveis à DMSO (a partir do processo de congelamento), meios de comunicação inicial pode ser substituído com a mídia fresco depois que as células tenham aderido à placa (aproximadamente quatro horas após a plating). Observe também que confluência das células após o descongelamento depende do número de células congeladas, a taxa de recuperação das células após o congelamento, ea taxa de crescimento das células. </li></ol> Células Passaging <ol start="5"><li> Quando as células têm crescido a confluência essencialmente 100% (ou seja: no dia seguinte), aspirar a mídia off e lavar as células com 5mL de PBS estéril. </li><li> Remover PBS e adicionar 1 ml de tripsina contendo 10mM EDTA e colocar a placa na incubadora por aproximadamente 5 minutos para permitir que as células de separar da placa. </li><li> Quando as células têm destacado, adicione 9ml de mídia para as células tripsinizados, pipeta 2-3 vezes para dispersar aglomerações, adicione 1 mL desta suspensão gota a gota, em um prato novo contendo 9ml de mídia, e colocar isso para a 37 ° C durante a noite incubadora . A presença de FBS na mídia vai inativar a tripsina, alternativamente, as células podem ser lavadas primeiro a diluir a tripsina. </li></ol> Contagem e cultivo de células em lamínulas <ol start="8"><li> Remover as células da incubadora, lavar e trypsinize como antes, só que desta vez adicionar 5-6ml de volta para o tripsinizados células em vez de 9ml. </li><li> Células pipeta cuidadosamente para garantir total dissociação em uma suspensão de células individuais, e remover 15μL para combinar com 15μL de azul de tripano em um tubo de microcentrífuga separado. </li><li> Misturar esta suspensão celular / azul de tripano pipetando cima e para baixo várias vezes, e adicionar 15μL para o hemocitômetro. </li><li> Sob o microscópio, as células mortas aparecerá azul. Contar o número de células claras / incolor (vivendo) em uma das grades 4X4 sobre o hemocitômetro. Repita este contar com as três outras grades 4X4 (A denotado, B, C e D) e use a seguinte equação para calcular o número de células por mL de suspensão: <img src="/files/ftp_upload/2160/2160eq0.jpg" alt="Equação 1" /></li><li> Através da otimização de protocolo, nós determinamos que chapeamento de 30.000 células resulta na densidade de células adequadas para visualização na conclusão do experimento. </li><li> Para calcular o volume de suspensão de células necessárias para 30.000 células, use a seguinte equação: <img src="/files/ftp_upload/2160/2160eq1.jpg" alt="Equação 2" /></li><li> Agora coloque uma lamínula estéril de vidro em cada poço de uma placa de seis bem e adicione 2 ml de mídia para cada poço, e depois adicionar gota a gota, a partir de sua suspensão celular o volume necessário para 30.000 células em cada poço. Note-se que lamínulas de vidro são usados ​​para este experimento, já que estaremos visualizando as células usando microscopia de fluorescência em um ponto mais tarde, nem todos os aplicativos serão necessariamente microscopia, e, assim, cobrir deslizes não podem ser exigidos. </li></ol> Transdução de vírus <ol start="15"><li> Depois de várias horas ou durante a noite (tempo suficiente para assegurar que as células aderem à lamínulas), remova a mídia e lavar as células com 1 ml de soro livre de DMEM. </li><li> Adicione 1 ml de soro livre de media a cada poço para a adição do vírus. Para este experimento, vírus adeno-Cre foi obtido comercialmente a partir de Biolabs Vector. </li><li> Otimização de protocolo anterior mostrou que uma multiplicidade de infecção (MOI) de 500 fornece transdução de genes altamente eficiente em MEFs primária com pouco ou nenhum efeito na viabilidade celular. MOI pode ser calculado como segue: <img src="/files/ftp_upload/2160/2160eq2.jpg" alt="Equação 3" /> <img src="/files/ftp_upload/2160/2160eq3.jpg" alt="Equação 4" /></li><li> Adicionar o volume calculado de vírus diretamente a cada poço que está a ser infectado, agite levemente o prato para dispersar as células de vírus e colocar na incubadora a 37 ° C durante a noite. </li><li> Na manhã seguinte, remova o vírus contendo mídia a partir de células e lavar as células em 1mL de PBS estéril e em seguida, adicione 2 mL de meios regulares a cada poço. </li><li> No caso do Rac1 células exemplificado neste experimento, ocorreram alterações morfológicas nas células em aproximadamente 72 horas de transdução de post. </li></ol> Resultados representante Células foram visualized através da coloração com o corante de actina phalloidin (para fins de imagem apenas) e fotografada usando a Leica TCS-SP5 multifotônica microscópio laser confocal no Centro de Análise Avançada da Universidade de Guelph. A Figura 1 mostra a morfologia contratados e alongado do Rac1 flox / flox células que foram expostos ao vírus adeno-Cre em comparação com as mesmas células não expostas ao vírus. <img src="/files/ftp_upload/2160/2160fig1.jpg" alt="Figura 1" /> Figura 1. Comparação entre Rac1 flox / flox células infectadas com o vírus adeno-Cre (esquerda) em comparação com células não infectadas pela mesma fonte (direita).

<ol>
	<li>Guo, F., Debidda, M., Yang, L., Williams, D. A., Zheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Deletion+of+Rac1+GTPase+Reveals+Its+Critical+Role+in+Actin+Stress+Fiber+Formation+and+Focal+Adhesion+Complex+Assembly.">Genetic Deletion of Rac1 GTPase Reveals Its Critical Role in Actin Stress Fiber Formation and Focal Adhesion Complex Assembly.</a> J. Biol. Chem. 281, 18652-18659 (2006).</li><li>Glogauer, M., Marchal, C. C., Zhu, F., Worku, A., Clausen, B. E., Foerster, I., Marks, P., GP, D. o. w. n. e. y., Dinauer, M., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1+deletion+in+mouse+neutrophils+has+selective+effects+on+neutrophil+functions.">Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions.</a> J. Immunol. 170, 5652-5657 (2003).</li><li>Prost, S., Sheahan, S., Rannie, D., Harrison, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+Cre+deletion+of+floxed+sequences+in+primary+mouse+cells+is+an+efficient+alternative+for+studies+of+gene+deletion.">Adenovirus-mediated Cre deletion of floxed sequences in primary mouse cells is an efficient alternative for studies of gene deletion.</a> Nucleic Acids Res. 29, E80-E80 (2001).</li><li>Rijnkels, M., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-Cre-mediated+recombination+in+mammary+epithelial+early+progenitor+cells.">Adenovirus-Cre-mediated recombination in mammary epithelial early progenitor cells.</a> J. Cell. Sci. 114, 3147-3153 (2001).</li><li>Vidali, L., Chen, F., Cicchetti, G., Ohta, Y., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1-null+mouse+embryonic+fibroblasts+are+motile+and+respond+to+platelet-derived+growth+factor.">Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor.</a> Mol. Biol. Cell. 17, 2377-2390 (2006).</li><li>Wang, H., Xie, H., Zhang, H., Das, S. K., Dey, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+recombination+by+adenovirus-driven+Cre+in+the+mouse+uterus.">Conditional gene recombination by adenovirus-driven Cre in the mouse uterus.</a> Genesis. 44, 51-56 (2006).</li></ol>

Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pelo Canadian Council on Animal Care.

O vídeo que acompanha exemplifica como um vírus recombinante pode ser usada para um gene especial de consumo específico in vitro utilizando Cre recombinase. O desenvolvimento deste protocolo no entanto revelou alguns pontos em particular no valor de endereçamento. <ul><li> Procedimentos de segurança deve sempre ser empregada quando se trabalha com adenovírus. Um jaleco e luvas devem ser usadas todo tempo enquanto trabalhava com o vírus. Mídia e utensílios de plástico devem ser tratadas com lixívia a 10% por pelo menos 30 minutos, e utensílios de plástico devem ser autoclavados depois. </li><li> Evite repetidos de congelamento e descongelamento do estoque de vírus como este podem afetar a título de vírus e, assim, reduzir a eficiência de transdução. Pequenos volumes de estoque de vírus deve inicialmente ser aliquotado em tubos separados. </li><li> MOIS variando 5-50 foram testados durante o desenvolvimento do método e as taxas de transdução eficiente foram observados na maioria dos casos, sem efeitos negativos sobre a viabilidade celular. </li><li> Adeno-GFP foi usado também durante o desenvolvimento do método para rapidamente a eficiência de transdução de tela. Este controle (ou um vetor vazio) deve ser realizada durante o experimento para assegurar que a infecção viral por si só não resulta em alterações celulares. Da mesma forma, se excisão do gene floxed de interesse em suas células não resultar em qualquer alteração celular (ou seja, alterações na morfologia ou viabilidade), então infectar o mesmo tipo de célula com adeno-GFP é um controle importante usar para demonstrar que a transdução de vírus é ocorrendo. </li></ul> Além do exame da cascata de sinalização na cultura, a abordagem adeno-Cre também tem sido empregada in vivo para condicionalmente remover genes de animais experimentais 3,4, e para rotular populações específicas de células dentro de um organismo 6. O sistema de adenovírus também pode ser adaptado para introduzir genes que não Cre recombinase em células hospedeiras. Duas principais vantagens do uso deste vetor de entrega são os seus transdução e eficiência de entrega de genes, e falta de integração com o DNA do hospedeiro. No entanto, a geração de novos adenovírus recombinante pode ser trabalhoso. O protocolo aqui descrito, portanto, utiliza a força do sistema adenoviral explorando o anteriormente desenvolvido vírus adeno-Cre, e acoplamento que com o nosso floxed alelo de interesse, Rac1. Através deste knock out condicional experimento, temos confirmado que transdução usando o vírus adeno-Cre de MEFs carregando um alelo Rac1 floxed realmente causar excisão de Rac1 levando à mudanças na morfologia celular característica de Rac1 deficiente células 2,5.

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<td>Ad-CMV-Cre virus</td>
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adenovirus-mediated genetic removal of signaling molecules in cultured primary mouse embryonic fibroblasts

A capacidade de remover componentes geneticamente específicos de diversas cascatas de sinalização celular tem sido uma ferramenta essencial na análise moderna de transdução de sinal. Um determinado método para alcançar essa supressão condicional é através da utilização do sistema Cre-loxP. Este método envolve flanqueiam o gene de interesse com sites loxP, que são sequências de reconhecimento específico para a proteína recombinase Cre. Exposição dos chamados floxed DNA (ladeada pelo site loxP) para este enzima resulta em um evento de recombinação Cre-mediada nos locais loxP, e excisão subseqüente do gene intervindo 3. Existem vários métodos diferentes para administrar Cre recombinase ao local de interesse. Neste vídeo, demonstramos a utilização de um adenovírus contendo o gene Cre recombinase para infectar fibroblastos de rato principal embrionárias (MEFs) obtidas de embriões que contém um alelo Rac1 floxed 1. Nossa justificativa para a seleção Rac1 MEFs para nossos experimentos é que claro alterações morfológicas podem ser vistos após a exclusão de Rac1, devido a alterações no citoesqueleto de actina 2,5. 72 horas após a transdução viral e expressão Cre, as células foram coradas com o corante phalloidin actina e fotografada por microscopia confocal de varredura a laser. Observou-se que MEFs que haviam sido expostos ao vírus adeno-Cre apareceu contratada e na morfologia alongada em comparação com células não infectadas, de acordo com relatórios anteriores 2,5. O método de adenovírus Cre entrega recombinase é vantajoso como o vírus adeno-Cre é facilmente disponível, e deleção do gene através de Cre em quase 100% das células pode ser alcançado com a infecção adenoviral otimizado.

Watch this Scientific Journal Video about Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts at JoVE.com

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

Neste vídeo, usamos um adenovírus carregando o gene Cre recombinase para infectar fibroblastos de rato principal embrionárias carregando um alelo Rac1 floxed.

Adenovírus mediada por remoção genética das moléculas de sinalização em fibroblastos de rato cultivadas Primária embrionárias

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

adenovirus-mediated-genetic-removal-signaling-molecules-cultured

Research

JoVE Journal

Biology

24.8K visualizações.  University of Guelph. Neste vídeo, usamos um adenovírus carregando o gene Cre recombinase para infectar fibroblastos de rato principal embrionárias carregando um alelo Rac1 floxed.

Vídeo: Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Imagens ao vivo de vesículas com núcleo denso na Primária Cultivadas neurônios do hipocampo

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Biologia

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Geração de células-tronco pluripotentes induzidas pela reprogramação fibroblastos de rato embrionárias com um fator de transcrição Quatro, Doxiciclina induzível Sistema de Transdução Lentivirus

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons2. This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate3,4 and the chemoattractive activities of Netrin15 and Sonic Hedgehog (Shh)6 in the floor plate in the spinal cord.
Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels7. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag18. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons3,4 as well the necessity for this signal to direct the commissural trajectory9.

This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.

Doseamento do Capacidade de difusíveis moléculas sinalizadoras para Reorientar embrionárias Spinal Axônios comissural

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo de imagens e tratamentos terapêuticos em um modelo de camundongo ortotópico de câncer de ovário

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

A remoção rápida de fibroblastos de Alta Densidade Humanos culturas de células estaminais embrionárias

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Um Sistema de<em&gt; Ex vivo</em&gt; A cultura de Pâncreas embrionárias

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Análise Genética rápida de epitelial-mesenquimal sinalização durante a regeneração do cabelo

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

Transporte Organelle em Cultivadas<em&gt; Drosophila</em&gt; Células: Linha celular S2 e primário neurônios.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Medição da captação de glicose e resposta à estimulação de insulina em miotubos primários humanos cultivados in vitro

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-interno pilha injeção maciça de células-tronco embrionárias leva a taxas mais elevadas de quimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...